Antibody against rat postacrosome reaction sperm and utilization thereof

ABSTRACT

An antibody biding specifically to rat&#39;s acrosome reacted sperm is produced and hybridomas (FARS-91 and FARS-92 strains) capable of stably proliferating are obtained by fusing mouse spleen cells having a high antibody titer against rat&#39;s acrosome reacted sperm with mouse-origin myeloma cells and screening fused cells reacting strongly with rat&#39;s acrosome reacted sperm. From these hybridomas, monoclonal antibodies selectively binding to rat&#39;s acrosome reacted sperm can be obtained. Thus, a diagnostic method for evaluating fertility of rat&#39;s spermatozoa is presented.

This application is a 35 U.S.C. 371 filing of PCT/JP00/07374 filed 23Oct. 2000.

TECHNICAL FIELD

The present invention relates to monoclonal antibodies selectivelybinding to rat acrosome reacted sperm, hybridomas producing saidmonoclonal antibodies, and a method for evaluating fertility of ratspermatozoa using said monoclonal antibodies. The invention also relatesto a method for screening materials effecting on rat fertility,characterized in using said method for evaluating fertility.

BACKGROUND ART

It is now obligated to do reproductive and developmental toxicitystudies in studies relating to safety of a drug on the occasion of anapplication of manufacturing approval for the drug. The reproductive anddevelopmental toxicity studies mean that animal experiments areconducted to obtain information on whether the application of a drug toliving bodies could possibly induce some adverse effect in the course ofreproduction and development. The experimental results can beextrapolated to humans and utilized in the evaluation of safety (risk)of the drug to reproduction and development in humans. A guideline forreproductive and developmental toxicity studies promulgated in 1997 wasrevised and enacted in conformity with the ICH guideline (InternationalConference on Harmonization of Technical Requirements for Registrationof Pharmaceuticals for Human Use; at the international conference forharmonization of regulation of pharmaceuticals in three areas involvingJapan, USA and EU) which was advised on 1994. In this guideline, it isdescribed that histopathological examinations of reproductive organs anda sperm analysis in repeated-dose toxicity studies should be maderelative to evaluation of male fertility in studies relating tofertility and early embryonic development to implantation.

To perform the histopathological examination for male reproductiveorgans, however, skillful technique is required, and there is sometimesa case given an effect to spermatozoa which could not be detected bymeans of the histopathological examination. In order to correctlyevaluate fertility and precisely examine an effect of a drug onspermatozoa, it is necessary to perform the fertility examination ofspermatozoa themselves as well as the histopathological examination.

In the current reproductive and developmental toxicity studies, noexamination of concentration, motility, morphology of spermatozoa andthe like that have been performed in human sperm analysis. Accordingly,an investigation for establishing a method for evaluation of spermatozoamotility is now being continued. This means that a method for evaluationof fertility has not yet been established.

As for examinations of fertility of human spermatozoa, a hamster test,conserved zona pellucida passage test, and triple-stain test and thelike have been developed. The hamster test is a method for evaluatingfertility, in which the properties of spermatozoa are utilized, that is,only spermatozoa which have the capacitation and in which the acrosomereaction have been completed, can enter the zona-free hamster eggs. Theconserved zona pellucida passage test is a method in which theproperties of spermatozoa are utilized, that is, only spermatozoa whichhave the capacitation, in which the acrosome reaction have beencompleted and in which the motility is retained sufficiently, can passthrough the conserved pellucid zone.

The triple-stain method is a method for distinguishing dead spermatozoa,live spermatozoa and spermatozoa of intact acrosome by means of stainingthem with a combination of three types of staining methods.

When the above-mentioned test that, so far, is known for humanspermatozoa is applied to the reproductive and developmental toxicitystudies, it is necessary to collect eggs to eliminate the zonapellucida, or to permits maturation of the collected immature eggs invitro and preserve them in a particular salt solution at a highconcentration to form the zona pellucida. These procedures, however,require skillful technique and troublesome operation as well asexpensive equipment. When the influence of chemical materials such asdrugs or environmental hormones on fertility of spermatozoa is examined,in the current reproductive and developmental toxicity studies thedecision whether or not to be fertilized on an experimental animal isnot determined from their appearance as long as the fetus has grown upto some degree after mating. This necessitates time and cost. Eventhough many points such as the number of spermatozoon, mobility andmorphology, which appear to have some influence on fertilization, areexamined, it is impossible to determine the exact fertility so long asthe presence of acrosome reaction as an essential factor forfertilization cannot be confirmed.

DISCLOSURE OF INVENTION

(Problems that the Invention is to Solve)

It is now necessitated, in the current reproductive and developmentaltoxicity studies, to develop an accurate and conveniently operablemethod for evaluating fertility of spermatozoa, in which the test can becompleted within a short period of time. It is also necessary that thetest results in the above method can be extrapolated to humans.

(Means for Solving the Problems)

The present inventors paid attention to utilizing an antigen-antibodyreaction in order to solve the above problems.

The present inventors have succeeded in preparing monoclonal antibodiesspecific to non-human animal spermatozoa post acrosome reaction as wellas hybridoma producing said monoclonal antibodies. At the same time,using these monoclonal antibodies, they established a method forevaluating fertility of spermatozoa, wherein the test results can beextrapolated to humans, and they also prepared a composition for use indetermining fertility. Moreover, they also established a method forscreening materials that affect fertility.

According to the invention, it is possible to complete the tests withina short period of time and to evaluate the fertility of spermatozoaaccurately in a convenient operation.

The principle for evaluating fertility in the invention is as follows.The spermatozoa just after ejaculation in mammals have no ability tofuse (fertilize) an egg. First, adsorptive materials such asglycoproteins and glycolipids contained in epididymis secretion orseminal fluid, which cover the surface of the protoplasmic membrane ofthe spermatozoa to protect the spermatozoa in female reproductiveorgans, are removed or denatured. This physiological alteration iscalled “capacitation”. Subsequently, the spermatozoa reaching theampulla of tubue uterinae, which is a spot for fertilization, make anapproach to an egg and cause an “acrosome reaction”. The acrosomereaction means a phenomenon in which the outermost protoplasmic membraneof spermatozoa fuses to the acrosomal outer membrane enveloping theoutside of the acrosome to open a small pore, through which an acrosomalenzyme is released from the acrosome. Only the spermatozoa that havecaused the acrosome reaction can fuse to the egg (fertilization) throughdissolution of the oval ambient pellucid zone by the acrosomal enzyme.Accordingly, the occurrence of the acrosome reaction is an essentialcondition for fertilization, and confirmation of the occurrence of theacrosome reaction is a direct method for evaluating fertility ofspermatozoa. The method of the invention, accordingly, can be used inevaluating the fertility of spermatozoa conducted in rats for thepurpose of the reproductive and developmental toxicity studies and thelike, on the assumption that the obtained test results can beextrapolated to humans.

In this connection, in order to carry out the evaluation of fertilityused in extrapolation to humans, it is necessary to choose experimentalanimals whose physiological state is very similar to that of humans.Until now, monoclonal antibodies to non-human animal acrosome reactedsperm have been reported on mice (Masaru Okabe et al., Journal ofReproductive Immunology, 11(2), 91-100, 1987), although as experimentalanimals used in extrapolation to humans, rats whose general metabolicpathways are well known have widely been used.

Therefore, monoclonal antibodies by which the occurrence of the acrosomereaction of rat spermatozoa can be distinguished were produced usingrats, an animal species which is the most studied in experimentalreproduction and development and whose physiological state is verysimilar to that of humans.

At present, regarding pharmaceuticals inhibiting the acrosome reactionof human spermatozoa, bicuculline (Calogero A. E. et al; Fertility andSterility, 71(5), 930-936, 1999), wortmannin (Fisher H. M. et al;Molecular Human Reproduction, 4(9), 849-855, 1998), cysteamine (SengokuK. et al; Journal of Andrology, 19(1), 37-49, 1998), anantin (Rotem R.et al; American Journal of Physiology, 274(2 Pt 1), E218-223, 1998),chlordane and endosulfan (Turner K. O. et al; Journal of Andrology,18(6), 571-575, 1997), etc., have been reported. In the future,monoclonal antibodies to acrosome reacted sperm will be useful forinvestigating the effect of drugs on the acrosome reaction ofspermatozoa in the reproductive and developmental toxicity studies.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A shows reactivity of the antibody FARS-91 with the controlspermatozoa (bright field; magnification of 100)

FIG. 1B shows reactivity of the antibody FARS-91 with the controlspermatozoa (FITC excitation; magnification of 100)

FIGS. 1A and B show the same field of vision.

FIG. 2 is a photomicrograph showing reactivity of the antibody FARS-91to acrosome reacted sperm (spermatozoa treated with A23187).

FIG. 2A shows reactivity of the antibody FARS91 with spermatozoa treatedwith A23187 (bright field; magnification of 100)

FIG. 2B shows reactivity of the antibody FARS-91 with spermatozoatreated with A23187 (FITC excitation;

-   -   magnification of 100)

FIGS. 2A and B show the same field of vision.

FIG. 3 is a photomicrograph showing reactivity of the antibody FARS-92to acrosome reacted sperm (spermatozoa as control treated with A23187).

FIG. 3A shows reactivity of the antibody FARS-92 with the controlspermatozoa (bright field; magnification of 100)

FIG. 3B shows reactivity of the antibody FARS92 with the controlspermatozoa (FITC excitation; magnification of 100)

FIGS. 3A and B show the same field of vision.

FIG. 4 is a photomicrograph showing reactivity of the antibody FARS-92to acrosome reacted sperm (spermatozoa treated with A23187)

FIG. 4A shows reactivity of the antibody FARS-92 with spermatozoatreated with A23187 (bright field; magnification of 100)

FIG. 4B shows reactivity of the antibody FARS-92 with spermatozoatreated with A23187 (FITC excitation; magnification of 100)

FIGS. 4A and B show the same field of vision.

FIG. 5 is a photomicrograph showing the frozen and thawed spermatozoaobserved by FITC-PSA staining.

FIG. 5A shows fresh spermatozoa (positive; magnification of 400)

FIG. 5B shows frozen and thawed spermatozoa (negative; magnification of400)

FIG. 6 is a photomicrograph showing reactivity of the antibody FARS-91with the frozen and thawed spermatozoa.

FIG. 6A shows reactivity of the antibody FARS-91 with fresh spermatozoa(negative; magnification of 200)

FIG. 6B shows reactivity of the antibody FARS-91 with the frozen andthawed spermatozoa (positive; magnification of 200)

FIG. 7 is a photomicrograph showing reactivity of the antibody FARS-92with the frozen and thawed spermatozoa.

FIG. 7A shows reactivity of the antibody FARS-92 with fresh spermatozoa(negative; magnification of 200)

FIG. 7B shows reactivity of the antibody FARS-92 with the frozen andthawed spermatozoa (positive; magnification of 200)

FIG. 8 is a photomicrograph showing reactivity of the antibody FARS-91with the frozen and thawed spermatozoa after treatment with A23187.

FIG. 8A shows reactivity of the antibody FARS-91 with the frozen andthawed spermatozoa after treatment with A23187 (bright field;magnification of 100)

FIG. 8B shows reactivity of the antibody FARS-91 with the frozen andthawed spermatozoa after treatment with A23187 (FITC excitation;magnification of 100)

FIGS. 8A and B show the same field of vision.

FIG. 9 is a photomicrograph showing reactivity of the antibody FARS-92with the frozen and thawed spermatozoa after treatment with A23187.

FIG. 9A shows reactivity of the antibody FARS-92 with the frozen andthawed spermatozoa after treatment with A23187 (bright field;magnification of 100)

FIG. 9B shows reactivity of the antibody FARS-92 with the frozen andthawed spermatozoa after treatment with A23187 (FITC excitation;magnification of 100).

FIGS. 9A and B show the same field of vision.

FIG. 10 is a photomicrograph showing reactivity of the antibodiesFARS-91 and FARS-92 with the frozen and thawed human spermatozoa.

FIG. 10A shows reactivity of the antibody FARS-91 with the frozen andthawed human spermatozoa (FITC excitation; magnification of 200)

FIG. 10B shows reactivity of the antibody FARS-92 with the frozen andthawed human spermatozoa (FITC excitation; magnification of 200)

BEST MODE FOR CARRYING OUT THE INVENTION

According to the invention, monoclonal antibodies to rat acrosomereacted sperm and two hybridomas, FARS-91 and FARS-92, producing saidantibodies were obtained. These hybridomas were deposited respectivelyin the Ministry of International Trade and Industry, National Instituteof Bioscience and Human-Technology (NIBH), located in 1—1-3, Higashi,Tsukuba-shi, Ibaragi, 305-8566, Japan, respectively, as accession nos.FERM BP-7401 and FERM BP-7402, on Oct. 13, 1999.

In producing the above antibodies, first, fresh rat spermatozoa, whichare collected by ejaculation or by removal of the cauda epididymis orseminal duct and which have not led to acrosome reaction (hereinafterreferred to as fresh rat spermatozoa), are provided and the acrosomereaction induced. Next, the rat acrosome reacted sperm serving asantigens are used to immunize mammals. Rat acrosome reacted sperm may beprepared by inducing the acrosome reaction by a chemical procedure, byinducing a pseudo acrosome reaction, by induction of the acrosomereaction in culture, or by a combination of these procedures and thelike.

The fresh spermatozoa may be obtained, for example, from the extirpatedcauda epididymis or seminal ducts. When the spermatozoa are collectedfrom the cauda epididymis, said cauda epididymis is cleaved into 3 or 4parts. When the seminal ducts are used, one duct is cut into 3 to 5portions. These are then immersed and gently swirled in a culture mediumsuch as TYH medium containing 0.4% BSA or m-KRB medium containing 0.5%BSA, at 30-40° C., for example, for 10 minutes, until the spermatozoamigrate. Alternatively, when collected from the cauda epididymis, thespermatozoa may be obtained by cutting a part of the seminiferous tubuleremoved from the cauda epididymis and scooping it up with around-pointed Pasteur pipette.

The chemical induction of the acrosome reaction is exemplified by amethod for treating fresh rat spermatozoa with a calcium ionophore suchas A23187. In addition, a method for treating with an anionicsurfactant, e.g., sodium deoxycholate, or a cationic, non-ionic orampholytic surfactant, may be employed.

The induction of a pseudo acrosome reaction may be achieved, forexample, by physically destroying the acrosomal outer membrane to exposethe acrosomal intima. Herein, this type of acrosome reaction is referredto as a “pseudo acrosome reaction”. The pseudo acrosome reaction may becarried out, for example, by freezing fresh rat spermatozoa at around−20° C. and then thawing. Alternatively, electroporation orultra-sonication may be utilized.

In a method for inducing acrosome reaction in culture, the method inwhich fresh rat spermatozoa are cultured in a medium containing calciumis exemplified. The concentration of calcium in the culture medium ispreferably set in a range of 0.5-5 mM, particularly at 1.8 mM. As asource of calcium, calcium chloride, calcium nitrate, calcium lactate,calcium gluconate, or the like may be used. As for the culture medium tobe used, media such as m-KRB, M-199, TYH and the like are included. TheTYH medium is composed of 6.976 g/L of sodium chloride, 0.356 g/L ofpotassium chloride, 0.251 g/L of calcium chloride dihydrate, 0.293 g/Lof magnesium sulfate heptahydrate, 0.162 g/L of potassium hydrogenphosphate, 2.160 g/L of sodium hydrogen carbonate, 0.111 g/L of sodiumpyruvate, 1.000 g/L of glucose, 4.0 g/L of bovine serum albumin, and 4.8mL of 50% syrup of sodium lactate. Additionally, an antibiotic oranti-fungal agent, e.g., penicillin, streptomycin, amphotericin B, etc,may be added to the medium.

Moreover, the above-mentioned methods for inducing acrosome reaction maybe used in combination. For example, a method comprising treatment withA23187 followed by freezing and thawing may be employed. Alternatively,fresh spermatozoa may be cultured in a medium containing calcium, duringwhich A23187 is also added. In such a case, the concentration of A23187is preferably added alone at a oncentration lower than that for inducingthe acrosome reaction.

Immunization may be performed on a mammal such as mouse, rat, and thelike. As a mammal, it is desirable to use an inbred animal strain withpermanently growing cells, which are used as a partner in cell fusion.Both of male and female animals may be used, preferably at the age of,for example, 3-10 weeks old. The number of rat spermatozoa used inimmunization, for example, for one mouse, is preferably 1×10³−1×10¹⁰.The spermatozoa are preferably mixed with, for example, Freund'scomplete adjuvant, Freund's incomplete adjuvant, aluminum adjuvant,pertussis adjuvant, endotoxins of gram-negative bacteria (Escherichiacoli, Salmonella, etc.), alum precipitate, clay grains (bentonite),aluminum compounds, oils, vitamins, plant polysaccharides, and the liketo form an emulsion, which is then administered to animalsintraperitoneally, intravenously, subcutaneously, or intracutaneously.The immunization may be done 1 to 5 times at intervals of 1-3 weeks. Inthe immunized animals, polyclonal antibodies to rat acrosome reactedsperm can be obtained from their body fluid or antibody-producing cellscontained in the body fluid. When the antibody titer has increasedsufficiently by measurement of the antibody titer of the animals, theantibodies or antibody-producing cells are collected.

In order to obtain monoclonal antibodies to rat acrosome reacted sperm,it is necessary to make hybridomas by fusing the antibody-producing cellwith a permanently growing cell. Preparation of hybridomas may becarried out as follows below.

The antibody-producing cells can be obtained from spleen, lymph node,peripheral blood, etc., with the spleen being preferred. The spleen ofthe immunized mammal is extirpated aseptically 2-5 days after the finalimmunization to make a suspension of spleen cells. As the permanentlygrowing cells for use as partners for fusion, cells which have beenknown by the person skilled in the art and which have a permanentlygrowing property can optionally be used, even though myeloma cells havewidely been used. It is preferable to use the permanently growing cellsderived from an animal homologous to that from which theantibody-producing cells have been obtained. In the case of mice,P3U1P3X63-Ag8.U1 (P3U1), P3/NS1/1-Ag4-1 (NS-1), SP2/0-Ag14 (SP-2),P3X63Ag8 (X63), P3X63-Ag8.653 (653), and the like may be used. As forthe permanently growing cells, it is preferable to use those having acharacteristic property utilizable as a marker in selection, forexample, 8-azaguanine resistant cell line, hypoxanthine guaninephosphoribosyl transferase-lacking cell line, etc. These cell lines canbe obtained from the American Type Culture Collection (ATCC).

For fusion, any one of these permanently growing cells is cultured in agrowth medium, then washed with, for example, a DMEM medium, andcollected by centrifugation prior to fusion. Fusion is achieved bymixing an antibody-producing cell with the permanently growing cell in aculture medium such as MEM, DMEM or RPMI1640, to which is added acell-fusing agent such as polyethylene glycol. If required, a smallquantity of dimethylsulfoxide may be added in order to accelerate thecell fusion. The resulting hybridoma is cultured in a FCS-containing MEMor RPMI1640 medium containing hypoxanthine, aminopterin, thymidine, etc.After a duration of about 1 week, the culture medium is changed to anFCS-containing MEM or RPMI1640 medium containing hypoxanthine andthymidine.

The hybridoma is then subjected to screening and cloning. In screening,the supernatant of hybridoma culture is collected and screened by aknown labeled antibody method, for example, radioimmunoassay, enzymeantibody method, fluorescent antibody method, etc., using rat acrosomereacted sperm as antigen. Subsequently, a population of hybridomasproducing a single monoclonal antibody is selected with cloning by aknown technique such as, for example, limiting dilution or soft agarmethod. It is preferred that screening and cloning be repeated two timesor more.

The hybridoma obtained as described above may be cultured in vitro (in aculture vessel or in a nutrition medium) or in vivo (in the living bodyor in animal tissue) to produce a monoclonal antibody. When cultured invitro, the hybridoma is cultured in a suitable medium such asFCS-containing MEM medium, RPMI1640 medium, etc., and the desiredmonoclonal antibody can be obtained from the supernatant of the culture.When cultured in vivo, the respective hybridomas are transplanted in theabdominal cavity of an animal that has a homologous histocompatibilityto an animal from which the permanently growing cell is derived, and areproliferated. Alternatively, the respective hybridomas are transplantedin nude mice, and the monoclonal antibodies produced in the ascites maybe collected. Prior to transplantation of the hybridoma, it is preferredto administer intraperitoneally a mineral oil such as 2, 6, 10,14-tetra-methylpentadecane (pristane).

The monoclonal antibodies contained in the culture supernatant or in thesupernatant of the ascites may be conventionally purified as known to aperson skilled in the art, although the antibodies can be used withoutpurification depending on the purpose. The purification may be achieved,for example, by means of salting out, such as by ammonium sulfateprecipitation, gel filtration with Sephadex, etc., dialysis with 0.02Mphosphate buffer (pH 7.2), etc., ion-exchange chromatography,electrophoresis, ultrafiltration, affinity chromatography, highperformance liquid chromatography, and the like.

In an embodiment of the invention, the antibodies of the invention canbe used in immunoassays such as immune staining, immune precipitation,immunoblotting, etc., for example, competitive or non-competitiveimmunoassay, radioimmunoassay, ELISA, latex aggregation, affinitycolumn, and the like. When ELISA is employed, a sandwich assay ispreferred. The immunoassay herein includes all methods utilizing immunereaction such as immunohistological examination, immunoblotting, immuneprecipitation, and the like. The antibodies of the invention can be usedin the collection and purification of rat acrosome reacted sperm sincethey can be utilized in the above-mentioned methods.

The antibodies of the invention can be used, for example, in evaluatingthe fertility of rat spermatozoa in the reproductive and developmentaltoxicity studies that relate to a safety study. In this method,spermatozoa to be evaluated are allowed to contact a labeled antibody ofthe invention, and the label attached to the spermatozoa is detected bya way of label detection. Alternatively, the spermatozoa are allowed tocontact an unlabeled antibody of the invention, which is then allowed tocontact a labeled second antibody (antibody capable of binding to theantibody of the invention). Thus, the label attached to the spermatozoais detected by means of label detection.

Labeling of antibodies may be effected, for example, with a fluorescentmaterial such as FITC (fluorescein isothio-cyanate) and RITC(tetramethylrhodamine isothiocyanate), a radioactive material such asradioactive iodine, radiocarbon, tritium, radium, strontium, etc., anenzyme such as peroxidase, an enzyme substrate, a coenzyme, an enzymeprecursor, an apoenzyme, a pigment, a chemiluminescent compound, aluminescent material, a color-producing material, a magnetic material,metal particles, and the like. Production of the second antibodies andattachment of a label to the antibodies may be achieved by aconventional means known to a person skilled in the art. A method fordetermining a fluorescent material includes, for example, a method ofobserving fluorescence excitation with ultraviolet rays under afluorescence microscope, a method of quantitatively determiningfluorescence intensity under irradiation of a certain excitation light,or the like. As a method for measuring radioactive materials, forexample, the amount of radiation can be measured using an α-rayspectrometer, a scintillation counter, an ionization box, a counter,etc., to determine the rat acrosome reacted sperm. As a method formeasuring an activity of peroxidase, for example, color development isconducted with o-phenylenediamine as a substrate, where the opticaldensity is measured to determine the rat acrosome reacted sperm.

As for a composition for measuring fertility using an antibody of theinvention, a composition in which the present antibody is kept, forexample, on beads (granules), is available. The beads on which theantibody is kept include carriers such as glass, Agarose, Sepharose,Agarose-loaded porous diatomaceous earth, hydrophilic copolymeric acrylgel, polystyrene, and the like. Preferably, superparamagnetic carriersin which a magnetizable material (e.g., Fe₂O₃) is contained in the coreare used. The shape of the granules is optional, including sphericalform, amorphous fractured form, etc., with a spherical form beingpreferred. There is no limitation in particle size, which may be, forexample, in a range of several micrometers to several hundredmicrometers.

Alternatively, it is also possible to examine fertility of ratspermatozoa, for example, by observing aggregation formed by the bindingof the spermatozoa to the above-mentioned antibody-carrying beads.Theoretically, the beads are cocultured with a certain number of freshspermatozoa in wells on a microplate for a certain period of time. Afterincubation of a certain period, the acrosome reaction starts to occurand binding of spermatozoa and beads is induced due to anantigen-antibody reaction. Further incubation leads to an increase inthe number of spermatozoa bound to beads due to an increase in thenumber of spermatozoa after induction of the acrosome reaction. Thebeads aggregate due to the motion of the bound spermatozoa. When acertain number of spermatozoa is cocultured together with a certainnumber of beads, the ability to induce the acrosome reaction can beestimated from the amount of spermatozoa bound to the beads. Though thestate of binding of spermatozoa to beads may be observed at any time, itmay be effected twice, 1-2 hours after incubation and 6-8 hours afterincubation. Thus, the rate of induction of the acrosome reaction can bedecided from the value after 1-2 hours. The cumulative number ofspermatozoa in which the acrosome reaction has occurred can be decidedfrom the value after 6-8 hours. In this state, when spermatozoa havingfertility are present at a certain number or more, multivalent bindingbetween granules and spermatozoa is induced and granules soon start toform aggregates, and granules or granule mass having no spermatozoabinding may disappear. Thus, it is possible to find the rate ofspermatozoa having fertility by examining the spermatozoa concentrationat which a certain number of beads can form aggregate.

Alternatively, the complex of spermatozoa and the aboveantibody-carrying beads (beads carrying a primary antibody) is gatheredwith a magnet in order to wash the complex, and then a secondaryantibody labeled with a fluorescent compound is added to determine thenumber of rat acrosome reacted sperm. The secondary antibodies may bethose which recognize rat spermatozoa or rat spermatozoa after theacrosome reaction.

When the secondary antibody recognizes rat acrosome reacted sperm, it isdesired that the epitope recognized by the secondary antibody isdifferent from that recognized by the primary one.

In order to practice the above test, it is convenient to make a kitcomprising materials necessary for carrying out the test. Such a kit maycontain granules for test, a plate having wells, a culture medium forincubating sepermatozoa and the like. The culture medium may containmineral salts, organic acid salts, sugars, serum albumin, antibiotics,indicators, and the like. In addition, the kit may contain test tubes,tubes for centrifugation, other similar glass vessels, pipettes orsimilar suction instruments, a microscope, and the like. In place of theabove granules for test, a kit may be included a good combination of theantibodies and the solid granules as raw material.

The plate equipped with wells for use in the invention may be made ofplastic, ceramics, glass, enamel, and the like. The bottom of the wellmay be in any shape such as flat, U- or V-shape, with a flat bottombeing preferred. The vertical section of the well is usually in acircular form. The number of the wells present on a plate, for example,96 wells, is optional. Observation is usually carried out under amicroscope, preferably at a magnification of 100-400.

More specifically, for example, a TYH culture broth containing no sodiumhydrogen carbonate and bovine serum albumin, 2-15% sodium hydrogencarbonate aqueous solution, lyophilized bovine serum albumin, FARS-91 orFARS-92 antibody-binding beads, and 20-70% glycerin solution are used.First, 1 mL of 7% sodium hydrogen carbonate solution is preferably addedto 22 mL of TYH culture broth and mixed. Thereafter, lyophilized bovineserum albumin is added thereto and dissolved without forming foam toprepare 0.4% bovine serum albumin-containing TYH culture broth. A vialcontaining the culture broth, of which the mouth was capped with aflame-sterilized aluminum foil, was equilibrated in a 5% carbon dioxidegas incubator at 37° C. for 1-18 hours, and may be used in a test. Inaddition, FARS-91 antibody or FARS-92 antibody-binding beads are mixedwell in a test tube mixer for about 30 seconds, of which 20 μL isimmediately distributed into 0.5 mL sterilized microtube, and 380 μL ofthe culture broth is then accurately added to the microtube. At thistime, the concentration of the beads may be adjusted to1.0×10³−1.0×10³10 particles/mL, preferably, 0.5×10⁶ particles/mL (beadsuspension). After adjustment, the mixture is preferably preserved in acold environment. 50% Glycerin solution may be added to a spermatozoasuspension in an amount equal to that of the spermatozoa suspension atthe time of counting spermatozoa in order to ease the counting.

Next, a test tube containing the culture broth is provided, to which isadded fresh rat spermatozoa. The mixture is then gently dispersed forseveral minutes, and the spermatozoa concentration is measured. It ispreferable to prepare a spermatozoa suspension at a concentration inwhich the spermatozoa number is 1×10⁶ particles/mL, using the culturebroth. At this time, 50% glycerin solution may be added to a spermatozoasuspension in order to ease the counting.

Next, a sterilized 96-well microplate is provided. The test can beachieved using 4 wells of the plate. That is, 100 μL of the culturebroth is added to the 2nd, 3rd and 4th wells, and the preparedspermatozoa suspension is added to the 1st and 2nd wells. The 2nd wellis agitated by means of pipetting, in which 100 μL is added to the 3rdwell. This same operation is repeated to the 4th well, from which 100 μLis removed. By means of this operation, 1-, 2-, 4 and 8-fold serialdilution can be made for the spermatozoa suspension. The 1st wellcorresponds to a 1-fold dilution, the 2nd to a 2-fold dilution, the 3rdto a 4-fold dilution, and the 4th to a 8-fold dilution. The beadsuspension is suspended on a mixer, in which 100 μL of each dilution isadded to each well. Immediately, each well is gently agitated with thetip of pipette in sequence from the 4th well to the 1st well. Themixture is then incubated without agitation at 37° C. in 5% carbondioxide gas. Then, the aggregate formed in the well is observed in orderto determine fertility.

ELISA (double layer method) may be carried out, for example, as follows.For example, rat spermatozoa which have been incubated for a period oftime sufficient to induce acrosome reaction are immobilized at thebottom of a well of a plastic plate, to which an antibody (primaryantibody) specific to the rat's acrosome reacted sperm is subsequentlyadded. Immobilization of the spermatozoa to be tested in the well may beachieved, for example, by immobilization with paraformaldehyde, etc.,and subsequently drying, for example, at 37° C. for about 24 hours. Ifthere are rat spermatozoa in which the acrosome reaction has occurred,an antibody will bind to them. Subsequently, after the wells are washed,for example, an enzyme-labeled second antibody is added to bind to theprimary antibody. This enzyme is allowed to react with a coloringsubstrate, and the absorbance is measured to determine the number of ratacrosome reacted sperm.

ELISA (sandwich method) may be carried out, for example, as follows. Anantibody (primary antibody) specific to the rat acrosome reacted spermis immobilized at the bottom of a well of a plastic plate, to which ratspermatozoa that have been incubated for a period of time sufficient toinduce the acrosome reaction are added. If there are rat spermatozoa inwhich the acrosome reaction has occurred, an antibody will bind to them.Subsequently, after the wells are washed, for example, afluorescently-labeled second antibody is added to bind to rat acrosomereacted sperm. The fluorescence intensity for this fluorescent substanceis measured to determine the number of rat acrosome reacted sperm. Inthis connection, the primary antibody and the second antibody may beused in a combination with either a monoclonal antibody or a polyclonalantibody. However, in a combination of a monoclonal antibody and anothermonoclonal antibody, it is preferable that the epitope of each antibodyis different from each other.

As a fluorescent antibody technique, for example, a method for directlylabeling an antibody to rat acrosome reacted sperm with a fluorescentsubstance (direct method), a method for labeling an antibody (secondantibody) recognizing the antibody to rat acrosome reacted sperm(indirect method), and a method for binding a second fluorescentlylabeled antibody through a complement, are included.

In a specific example of the indirect method, fresh rat spermatozoa arediluted with 0.1%-5.0%, preferably 0.4%-2.0% BSA-containing TYH culturebroth to prepare a mixture at a concentration of 1.0×10³sperms/mL−1.0×10¹⁰ sperms/mL, preferably 0.2×10⁶ sperms/mL−1.5×10⁶sperms/mL. Next, the mixture is incubated for a period of timesufficient for inducing the acrosome reaction, then an antibody to ratacrosome reacted sperm is added, and allowed to react at 30-40° C. for5-60 minutes. The reaction mixture is washed with the culture broth bycentrifugation, an FITC-labeled anti-mouse IgG is added, and allowed toreact at 30-40° C. for 5-60, minutes. After the reaction is complete,the mixture is washed several times with 0.001-0.5% NaN₃-containing PBS(Ca, Mg free) by centrifugation, and observed under a fluorescencemicroscope. Thus, the rate of spermatozoa for which fluorescence isrecognized at the head can be determined.

Rat spermatozoa used as specimen may be in a state of seminal fluidwithout processing such as by purification or may be in a state of livespermatozoa isolated by swimming-up, or in a state in which thereactivity with an antibody induced by addition of an agent such asprotease to seminal fluid (spermatozoa). The specimen may be in a stateof suspension in the culture broth, or in a state of being fixed orimmobilized in a plate or dish made of glass or plastic, or in a stateof fixation with paraformaldehyde. Fixation with para-formaldehyde maypreferably be carried out at a concentration of 0.5-15, preferably3%-10%.

This antibody can be used in an in vivo screening test of substancesthat possibly influence the fertility of rat spermatozoa, such as forexample, drugs, environmental hormones, etc. The screening test may becarried out, for example, as follows. For example, a substance to betested is administered, e.g., orally, intravenously, intraarterially,subcutaneously, intramuscularly, intraperitoneally or applied locally(to skin, as eye drops or ear drops, nebula, suppositories, etc.) to amatured week old male rat once a day for a sufficient period requiredfor the whole process of spermatogenesis and maturation of spermatozoa.Then, the spermatozoa are collected, for example, from the caudaepididymis. The collected spermatozoa are incubated, for example, for asufficient period required for the acrosome reaction, and theirfertility is determined by an indirect fluorescent antibody method.

This antibody can also be used in an in vitro screening test ofsubstances possibly influencing the fertility of rat spermatozoa. Thescreening test may be carried out, for example, as follows. For example,a substance to be tested such as a drug is added to the spermatozoacollected, for example from the cauda epididymis on a fully matured weekold male rat, and incubated. Then, after incubation for several minutesto several hours, for example, the fertility is determined by anindirect fluorescent antibody method.

EXAMPLES

The following examples serve to illustrate the invention for furtherunderstanding, but they should not be interpreted to limit the technicalscope of the invention.

Example 1 Method for Collecting Immunogens

Wistar male rats (age in 14 weeks) were killed by exsanguination underanesthesia with ether, from which the cauda epididymides were removedand immediately placed in a Petri dish containing 10 mL of m-KRB(modified Krebs-Ringer bicarbonate medium) containing 1% BSA (bovineserum albumin; Bayer) warmed at 37° C. (1 plate per cauda epididymis).Cleavages were made at 2-3 parts of the cauda epididymis, which wasgently stirred and allowed to stand for about 5 minutes to releasespermatozoa. The spermatozoa suspension is moved into a 50 mL tube, intowhich a calcium ionophore A23187 (Sigma) was added thereto at a finalconcentration of 10 μM and incubated for about 15 hours in a CO₂incubator. After terminating incubation, the treated spermatozoa werewashed twice with PBS(−), then adjusted at a predeterminedconcentration, and preserved at −20° C.

Example 2 Immunization of Mice

In Jcl female mice (age in 6 weeks), the first day of immunization wasregarded as 0 day, and booster immunization was made on the 14th, 21stand 28th days. For immunization, the above-treated rat spermatozoa wereadministered subcutaneously in the back of the mice at a dose of about1×10⁷ per shot as an equal mixture with Freund's complete adjuvant andas an equal mixture with Freund's incomplete adjuvant, respectively, onday 0 and on the 14th day. On the 21st and 28th days, only spermatozoawere administered intraperitoneally.

Example 3 Method for Preparation of Hybridoma

Three days after the final immunization, the spleen was removed from theimmunized mice and used in cell fusion.

A suspension of splenic cells prepared on an RPMI1640 medium (GIBCO) wasmixed with SP-2 cells (murine myeloma cells) at a ratio of about 4:1,permitted to undergo cell-fusion using polyethylene glycol 2000 (Sigma)and then washed with an RPMI1640 medium. Thereafter, the number of cellswas adjusted to be about 2×10⁶ cells/mL by dilution with an RPMI1640medium containing 15% FCS (fetal calf serum; Mitsubishi Chemical), ofwhich 100 μL each was distributed into each well of 96-well microplates,with 6 plates per mouse. The next day, 100 μL of HAT medium(hypoxanthine, aminopterin, thymidine, 15% FCS-containing RPMI1640medium) was added to each well. On the 2nd, 3rd, 5th and 8th days, halfof the medium was replaced with a HAT medium, and on the 10th and 13thdays, half of the medium was replaced with an HT medium (hypoxanthine,thymidine, 15% FCS-containing RPMI1640 medium).

Example 4 Screening by ELISA

After treatment with A23187, the frozen-thawed spermatozoa were preparedto 1×10⁶ cells/mL, of which 50 μL each was distributed into each well ofa 96-well flat-bottom microplate and dried at 37° C. for about 24 hoursfor immobilization of the spermatozoa. After immobilization, the platewas blocked with 250 μL of 2% BSA-containing PBS(−), sealed andpreserved at 4° C. before use. The plate was used within 5 days afterpreparation.

The blocking solution in sperm-immobilized plates was washed out, and 50μL each of the supernatant taken from each well of negative control(20-fold diluted normal murine serum) and positive control (1000-folddiluted spermatozoa-immunized murine serum) as well as 50 μL of thesupernatant of cell culture taken from each well 13 days afterincubation was distributed in each well and allowed to react at 37° C.for 2 hours. After the reaction was complete, the plates were washedtwice with 0.05% Tween 20-containing PBS(−), 50 μL/well ofperoxidase-labeled anti-mouse IgG (TAGO), which was one hundred thousandfold-diluted, was added, and allowed to react at room temperature for 2hours. The plates were then washed in the same manner, 100 μL/well of asubstrate solution (60 mg/dL of o-phenylenediamine solution, with 0.006%H₂O₂ added) was added, and allowed to react under shading at 37° C. forabout 20 minutes. Thereafter, the reaction was terminated with additionof 0.5M sulfuric acid (50 μL/well), and absorbance was measured with amicroplate reader (490 nm-650 nm). The specimen exhibiting theabsorbance equal to or over that of the positive control was judged tobe positive.

Example 5 Screening by an Indirect Fluorescent Antibody Method

After treatment with A23187, the frozen-thawed spermatozoa were preparedto 1×10⁶ cells/mL, where, to 20 μL of the cell suspension 50 μL each ofnegative control (20-fold diluted normal murine serum) and positivecontrol (1000-fold diluted spermatozoa-immunized murine serum) as wellas 50 μL of the cell culture supernatant taken from each well which waspositive in screening by the above ELISAs on the 14th day afterincubation. The mixture was gently stirred and allowed to react at 4° C.overnight or at 37° C. for 2 hours. After the reaction was complete, themixture was washed twice with 0.02% NaN₃-containing PBS(−), and 20 μL of400-fold diluted FITC-labeled anti-mouse IgG was added, gently stirred,and allowed to react at 37° C. for 1 hour. After the reaction wascomplete, the mixture was washed in the same manner, and observed undera fluorescence microscope. The presence of fluorescence recognized atthe head of spermatozoon was judged to be positive.

Example 6 Cloning of Fused Cells

Cloning was performed by means of limiting dilution.

The cells of the positive wells were prepared to a cell concentration of5-100 cells/mL with a cloning medium (HT medium containing 10% breiclone®), of which 100 μL each was distributed to each well of a 96-wellmicroplate. The cloning medium (100 μL) was then immediately added toeach well.

For the cells after cloning, the culture medium was changed at a rate of2-3 times per week (half was replaced with a fresh cloning medium), andscreening was performed in conformity with the growth of the cells.Repetition of screening and cloning established hybridomas FARS-91 andFARS-92.

Hereinafter, a monoclonal antibody produced by a mouse—mouse fused cellFARS-91 is referred to as FARS-91 antibody, and a monoclonal antibodyproduced by a mouse—mouse fused cell FARS-92 as FARS-92 antibody.

Example 7 Determination of Subclass of FARS-91 and FARS-92 Antibodies

Using a mouse monoclonal antibody isotyping kit (Amersham), thesub-class of antibodies was identified.

Three mL of ascites fluid containing FARS-91 antibody or FARS-92antibody was allowed to react with a typing stick at room temperaturefor 15 minutes, and then the typing stick was washed 3 times with 5 mLof Tris buffer saline. 3 mL of peroxidase-labeled anti-mouse antibodywas then added, allowed to react at room temperature for 15 minutes, andwashed in the same manner. Afterwards, 3 mL of a substrate solution wasadded, allowed to react at room temperature for 15 minutes, and thetyping stick was washed 3 times with distilled water. After drying, thesub-class of the antibody was identified by a sign observed on thestick.

As results, it was confirmed that the sub-class of the FARS-91 antibodyis IgG, and that of the FARS-92 antibody is IgG_(2b).

Example 8 Reactivity of FARS-91 and FARS-92 Antibodies to AcrosomeReacted Sperm

As described above, fertility of spermatozoa can be determined byconfirming whether the acrosome reaction has occurred in thespermatozoa. Accordingly, it was examined whether the monoclonalantibodies obtained in Example 6 can be utilized in the above-mentionedconfirmation test.

It has been reported that treating fresh spermatozoa with a calciumionophore A23187 causes an acrosome reaction in mice (Lynn R. Fraser;Journal of Andrology, 3, 412-419, 1982) and humans (Green et al.;Journal of Cell Science, 32, 321, 1978).

Therefore, the reactivity of FARS-91 antibody and FARS-92 antibody tospermatozoa treated with A23187 (acrosome reacted sperm) and to freshspermatozoa in rats was examined by an indirect fluorescent antibodymethod.

Spermatozoa collected from the cauda epididymis of Jcl:SD male rats wereprepared to 1×10⁶/mL with 0.4% BSA-containing TYH culture broth. A23187dissolved in DMSO was added to a final concentration of 2 μM, and themixture was allowed to react at 37° C. for 20 minutes. As a control,DMSO used as a solvent for A23187 was added and allowed to react in thesame manner. After the reaction was complete, the ascites fluidcontaining FARS-91 antibody or FARS-92 antibody was added, and allowedto react at 37° C. for 30 minutes. The mixture was washed twice with0.4% BSA-containing TYH culture broth by centrifugation (37° C., 2800rpm, 5 min), FITC-labeled anti-mouse IgG was added, and allowed to reactat 37° C. for 60 minutes. After the reaction was complete, the mixturewas washed twice with 0.02% NaN₃-containing PBS(−) by centrifugation(room temperature, 2800 rpm, 5 min), and observed under a fluorescencemicroscope to measure the rate of spermatozoa exhibiting fluorescence ontheir head.

As a result, it was found that 65.5±3.7% of spermatozoa treated withA23187 (FIGS. 2A and B) reacted with FARS-91 antibody (in controlspermatozoa (FIGS. 1A and B), 12.5±3.1%), while 59.8±3.8% of spermatozoatreated with A23187 (FIGS. 4A and B) reacted with FARS-92 antibody (incontrol spermatozoa (FIGS. 3A and B), 8.5+1.7%). From these results, itwas confirmed that the FARS-91 antibody and the FARS-92 antibody reactspecifically with spermatozoa in which the acrosome reaction hasoccurred.

Example 9 Observation of Frozen-Thawed Spermatozoa by a FITC-PSAStaining Method

It has been said that freezing and thawing of spermatozoa physicallydestroys the plasma membrane and/or acrosomal outer membrane and leadsto a state which follows the occurrence of the acrosome reaction(hereinafter, referred to as pseudo-acrosome reaction). Therefore, usingthe fluorescent pigment FITC-PSA which has specificity to the contentsof acrosomal granules present in the acrosome before the acrosomereaction, it was confirmed whether frozen-thawed spermatozoa cause thepseudo-acrosome reaction. In general, in spermatozoa in which noacrosome reaction occurs, fluorescence is observed at the sperm headsince the fluorescent pigment FITC-PSA is bound to the contents ofacrosomal granules in the acrosome.

Smears of fresh spermatozoa and frozen-thawed spermatozoa were prepared,allowed to stand at room temperature for 2 hours or longer to dry, fixedin 95% methanol for 2 hours, and washed 3 times by immersion indistilled water for every 10 minutes. After washing, the smears wereallowed to react with a FITC-PSA (50 mg/mL) solution at room temperatureunder shading for 3 hours. After washing with distilled water, thesmears were observed under a fluorescence microscope. Spermatozoaexhibiting fluorescence on their head were judged positive.

Fluorescence was observed on the head of fresh spermatozoa (FIG. 5A),while almost no fluorescence was observed on that of frozen-thawedspermatozoa (FIG. 5B).

That is, it was confirmed that fresh spermatozoa cause no acrosomereaction, but in frozen-thawed spermatozoa a pseudo-acromal reactiondoes occur.

Example 10 Reactivity of FARS-91 and FARS-92 Antibodies to Frozen-ThawedSpermatozoa

Since it was proven in Example 9 that a pseudo-acromal reaction occursin frozen-thawed spermatozoa, it was examined whether FARS-91 andFARS-92 antibodies react specifically with spermatozoa that cause thepseudo-acrosome reaction, i.e., frozen-thawed spermatozoa.

To 30 μL of fresh spermatozoa or frozen-thawed spermatozoa 1×10⁶/mL), 50μL of ascites fluid containing FARS-91 antibody or FARS-92 antibody wasadded. The mixture was gently stirred, allowed to react at 37° C. for 30minutes, and washed with 0.4% BSA-containing TYH culture broth bycentrifugation (37° C., 2800 rpm, 5 min). After washing, 30 μL of400-fold diluted FITC-labeled anti-mouse IgG was added and allowed toreact at 37° C. for 1 hour. The mixture was washed twice with 0.02%NaN₃-containing PBS(−) by centrifugation (room temperature, 2800 rpm, 5min) and observed under a fluorescence microscope.

As a result, fluorescence was observed at the head of most of thefrozen-thawed spermatozoa for both of the FARS-91 and FARS-92 antibodies(FIG. 6B and FIG. 7B), while the rate of fluorescent spermatozoa wasgreatly reduced in fresh spermatozoa (FIG. 6A and FIG. 7A). According toExamples 9 and 10, it was confirmed that the FARS-91 and FARS-92antibodies also react specifically with spermatozoa which cause apseudo-acrosome reaction.

Example 11 Reactivity of FARS-91 and FARS-92 Antibodies to Spermatozoawhich Have Been Frozen and Thawed After Treatment with A23187

Reactivity of the FARS-91 and FARS-92 antibodies was examined forspermatozoa which have been frozen and thawed after treatment withA23187 to cause chemical and physical acrosome reactions.

Spermatozoa collected from the cauda epididymis of Jcl:SD male rats wereprepared to 1×10⁶/mL with 0.4% BSA-containing TYH culture broth. A23187dissolved in DMSO was added to a final concentration of 2 μM, and themixture was allowed to react at 37° C. for 20 minutes. After thereaction was complete, the treated spermatozoa were washed twice withPBS(−), prepared to a predetermined concentration, and frozen at −20° C.After thawing, the ascites fluid containing FARS-91 antibody or FARS-92antibody was added, and allowed to react at 37° C. for 30 minutes. Themixture was washed twice with 0.4% BSA-containing TYH culture broth bycentrifugation (37° C., 2800 rpm, 5 min), 30 μL of FITC-labeledanti-mouse IgG was added, and allowed to react at 37° C. for 60 minutes.The mixture was washed twice with 0.02% NaN₃-containing PBS(−) bycentrifugation (room temperature, 2800 rpm, 5 min), and observed under afluorescence microscope.

As a result, fluorescence was observed on the head of nearly 100% ofspermatozoa which have been frozen-thawed after treatment with A23187 inthe FARS-91 (FIGS. 8A and B) and FARS-92 (FIGS. 9A and B) antibodies.

Example 12 Reactivity of FARS-91 and FARS-92 Antibodies to HumanFrozen-Thawed Spermatozoa

It was examined by an indirect fluorescence antibody method whether ornot the FARS-91 and FARS-92 antibodies react to human frozen-thawedspermatozoa.

To 30 μL of human frozen and thawed spermatozoa 1×10⁶/mL), 50 μL ofascites fluid containing FARS-91 antibody or FARS-92 antibody was added.The mixture is allowed to react at 37° C. for 30 minutes, and washedtwice with 0.02% NaN₃-containing PBS(−) by centrifugation (37° C., 2800rpm, 5 min). Then, 30 μL of 400-fold diluted FITC-labeled anti-mouse IgGwas added and allowed to react at 37° C. for 1 hour. The mixture waswashed in the same manner and observed under a fluorescence microscope.

Both of the FARS-91 (FIG. 10A) and FARS-92 (FIG. 10B) antibodies did notemit fluorescence in reaction with human frozen and thawed spermatozoa.Thus, it was shown that these antibodies are not bound to human frozenand thawed spermatozoa.

Example 13 Preparation of a Kit for Characterization of Fertility

A suspension of Dynabeads M-450 sheep anti-mouse IgG (FC) was wellshaken, and a necessary amount was placed on tubes for centrifugation ina clean bench and subjected to centrifugation at 4° C. and 2000 rpm for5 minutes. After removal of the supernatant, a non-specific adsorptionblocker, Block Ace (Snow Brand), was added in an amount equal to thecollected Dynabeads, and the mixture was incubated under shaking at 37°C. for 1 hour. After termination of incubation, the mixture wascentrifuged at 4° C. and 2000 rpm for 5 minutes to remove thesupernatant. Sterilized PBS(−) at twice the amount of the collectedDynabeads was added, and the mixture was stirred and centrifuged at 4°C. and 2000 rpm for 5 minutes to remove the supernatant. This operationwas further repeated twice to completely remove the non-specificadsorption blocker. Finally, PBS(−) in an amount equal to the collectedDynbeads was added to the precipitate, and then the ascites liquidcontaining the FARS-91 antibody or FARS-92 antibody was added. Themixture was mixed well and allowed to react under shaking at 37° C. for1 hour. Because dispersion of beads was insufficient under shaking dueto their heavy specific gravity, the mixture was frequently stirred wellusing a mixer during the reaction. After the reaction was complete, themixture was centrifuged at 4° C. and 2000 rpm for 5 minutes to removethe supernatant. Then, the sterilized PBS(−), in an twice amount of thecollected Dynbeads, was added to the precipitate. After mixing well, themixture was centrifuged at 4° C. and 2000 rpm for 5 minutes to removethe supernatant. This washing operation was repeated 3 more times tocompletely remove the FARS-91 or FARS-92 antibody that remain unchanged.Finally, PBS(−) containing 0.02% NaN₃ and 2% bovine serum albumin(BSA)in an amount that is 10 times the amount of the collected Dynabeads wasadded to the precipitate obtained. This bead suspension was mixed well,of which 20 μL was dispensed into microtubes, and to which microtubes380 μL of 2% BSA-containing PBS(−) was added. The number of beads wasprepared to be 0.5×10⁶/mL by counting on a Thoma's hemocytometer. On theother hand, spermatozoa collected from the cauda epididymis of Jcl:SDmale rats were prepared to be 1×10⁶/mL with 0.4% BSA-containing TYHculture broth. To 10 μL of antibody-binding beads at 0.5×10⁶/mL, 100 μLof fresh rat spermatozoa prepared to 1×10⁶/mL was added, and the mixturewas allowed to react at 37° C. under 5% CO₂.

For both the FARS-91 and FARS-92 antibodies, the aggregation of thebeads was not observed immediately after addition of rat spermatozoa,but it was recognized after a lapse of 6 hours.

Example 14 (Kit)

By combining the following components, a kit was prepared:

-   -   TYH solution containing no sodium hydrogen carbonate and bovine        serum albumin;    -   7% Sodium hydrogen carbonate;    -   Bovine serum albumin; and

FARS-91 beads or FARS-92 beads.

INDUSTRIAL APPLICABILITY

The present invention provides monoclonal antibodies reactingspecifically to rat acrosome reacted sperm. Additionally, the inventionprovides a kit for evaluation of fertility using said antibodies, bywhich fertility can be conveniently evaluated within a short period oftime without requiring skillful technique or troublesome operations.

1. A hybridoma deposited under accession number FERM BP-7401.
 2. Ahybridoma deposited under accession number FERM BP-7402.
 3. A monoclonalantibody specifically reacting to rat acrosome reacted sperm, saidmonoclonal antibody provided by a hybridoma deposited under accessionnumber FERM BP-7401.
 4. A method for evaluating fertility of ratspermatozoa, comprising: a) contacting rat spermatozoa to be evaluatedwith the antibody of claim 3, wherein said antibody is labeled; and b)detecting an amount of label attached to the spermatozoa and evaluatingthe fertility of rat spermatozoa.
 5. A composition for measuringfertility of rat spermatozoa, comprising the antibody of claim
 3. 6. Acomposition for measuring fertility of rat spermatozoa, comprising beadscarrying the antibody of claim
 3. 7. A monoclonal antibody specificallyreacting to rat acrosome reacted sperm, said monoclonal antibodyproduced by a hybridoma deposited under accession number FERM BP-7402.8. A method for evaluating fertility of rat spermatozoa, comprising: a)contacting rat spermatozoa to be evaluated with the antibody of claim 7,wherein said antibody is labeled; and b) detecting an amount of labelattached to the spermatozoa and evaluating the fertility of ratspermatozoa.
 9. A composition for measuring fertility of ratspermatozoa, comprising the antibody of claim
 7. 10. A composition formeasuring fertility of rat spermatozoa, comprising beads carrying theantibody of claim
 7. 11. A kit for evaluating fertility, comprising theantibody of claim 3 or claim
 7. 12. A kit for evaluating fertility,comprising the composition of any one of claims 5, 6, 9, and 10.